The microenvironment of tumors can be a hostile place, with significant regions that are hypoxic and acidic. The focus of this research program is on the tumor pH, although studies of tumor oxygenation are also included. Renewal funding is sought to continue improving methods for imaging tumor pH (Aim 1), and.to understand the causes (Aim 2) and consequences (Aim 3) of acid pile in this hostile microenvironment. During the last period of support we made significant advances in all three aims. New methods have been developed and exported, a fundamental relationship between aerobic glycolysis and hyperacidosis was uncovered, and the effect of acid pile in conferring resistance to ionizable drugs was characterized. From these last findings, a clinical trial is being planned to enhance the efficacy ofmitoxantrone therapy with metabolic alkalinization. One book and 27 manuscripts have been published, are in press or have been submitted. In Aim 1 of the next period of support, we propose to develop an exciting new class of MRI-sensitive Gd-containing pH reporters which hold promise for imaging pH with very high spatio-temporal resolution. A significant effort will be spent developing quantitative methods that would eventually be appropriate for the clinic. Experiments in aims 2 & 3 will focus on testing a novel model which treats the hypoxia and acidity in tumors as selective pressures that drive the evolution of the tumor phenotype from benign to metastatic. The model proposes that there are functional connections between transient hypoxia --_ dysregulation of the hypoxia response element, HIF-la _ aerobic glycolysis _ hyperacidity _ proteases --_ extravasation survival during colonization. Each of these connections has been observed in the literature, but has not been tested in a single system. Such a model could explain why elevated uptake of flurodeoxyglucose is commonly observed in metastatic cancers. Aim 2 will determine if there are functional connections between HIF-1 or, elevated aerobic glycolysis, metastatic potential and hyperacidic pile in tumors using primary and established human breast cancer cells. Aim 3 will determine if there are functional connections between glycolysis, acid pile and metastasis at the levels of extravasation and colonization. Although specific hypotheses will be tested, experiments are designed to yield important information even if the hypotheses are not true.